Phenethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study:

A reproduction/developmental toxicity screening test (OECD TG 422) was conducted on male and female Wistar rats to assess the reproductive performance and offspring development following oral administration of the test chemical. Groups of 13 rats/sex/dose were orally administered with 0 (vehicle control, corn oil), 308, 556 and 1000 mg/kg of test chemical. Groups of 5 rats/sex were administered either 0 or 1000 mg/kg test chemical daily till the first scheduled sacrifice and kept for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. All animals of both sexes were dosed 2 weeks prior to mating and during the mating period. Males were further dosed till day 47th day. Female rats were dosed during pregnancy till 4 day of postpartum. The oestrus cycle was monitored daily from the beginning of treatment until the evidence of mating. All animals were observed for mortality and morbidity twice daily. General clinical observations of animals in all groups were made daily, detailed clinical examination was performed once before the first treatment, then weekly. Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for 5 males and 5 females of each groups during the last week of treatment and for recovery groups during the last recovery week. The body weight and feed consumption were weighted weekly for both males and females. Pregnant females were weighted on day 0, 7, 14 and 20, post-partum day 0-1, on day 4 post-partum and before sacrifice. Haematology and clinical biochemistry were performed on 5 males and 5 females of each group, prior to necropsy and organs weights measurements. On termination day, all animals were subjected to gross pathological examination, ovaries, uterus, cervix with vagina, testes, epididymis, prostate, seminal vesicle with coagulation gland were preserved. Full histopathology of the preserved sexual organs of all animals and all tissues of five males and five females randomly selected from control and high-dose group. No mortality and morbidity were observed in both treatment and recovery groups during the entire study period. No apparent treatment-related clinical sign was observed in the treatment and recovery groups. Sensory reactivity and motor activity measurements were comparable, and no treatment related changes were revealed at any dose level when compared to control groups. No treatment-related and dose-dependent changes in mean body weight were noted up to 1000 mg/kg both in treatment and recovery groups. However, the mean body weight change (%) of males decreased significantly during the entire treatment period (Day 1-46) at 1000 mg/kg and during the first two weeks of treatment at 556 mg/kg. Males from the recovery group also demonstrated significant decrease in the mean body weight changes during day 1-29. Females gained significantly less weight during gestation (days 1-20), but the mean body weight change did not alter in the recovery group. The feed consumption of all treatment and recovery groups of both sexes were comparable and did not show any significant differences as compared with respective controls. In addition, no treatment related and/or dose dependent changes were noted in haematological and clinical biochemistry parameters up to 1000 mg/kg body weight. External and visceral examination of all treated and control rats including recovery groups did not reveal any abnormality of pathological significance. The absolute and relative organ weights of brain, adrenals, ovaries with oviduct, testes, epididymis, heart, liver, kidney spleen, thymus of dosed rats of either sex did not differ significantly when compared to respective control groups. Oestrus cyclicity was regular i.e. 3-5 days in control, treatment and recovery groups. During cohabitation, all females from treatment and control groups showed evidence of copulation. Most females showed pre-coital interval less than 5 days, except 4 females from the 1000 mg/kg, 1-1 females from 556 mg/kg and control groups which showed pre-coital interval longer than 5 days. Only 8 out of 13 females achieved pregnancy in the high-dose group and the pregnancy/fertility index decreased to 61.54%. The gestation length was comparable in all treatment and control groups. Examination of the uterus content revealed that the number of implantation and corpora lutea did not differ in treated and control rats. The reproductive organs of all dosed rats were subjected to histopathological evaluation. Histopathological lesions observed in males included focal minimal to mild seminiferous tubule degeneration, focal minimal to mild and multifocal minimal retention of mature sperm, focal minimal to mild sloughing of round spermatid/pachytene spermatocyte. Histopathological lesions observed in females included minimal reduction of stromal cells, focal moderate necrosis and nodular hyperplasia of uterus as well as minimal lymphocytic infiltration of the cervix. However, the severity and incidence of histopathological lesions of testis, uterus and cervix were well comparable with respective control groups and exhibited no dose relationship. Hence, the occurrence of these lesions was considered spontaneous or incidental in nature and did not attributed to the test chemical administration. Developmental parameters such as number of live births, litter size, number of alive pups at PND 4, and post-implantation loss were comparable in all groups. Postnatal loss (%) were increased at 556 and 1000 mg/kg but was not statistically significant. The fetal survival index at PND 4 slightly decreased in middle and top dose groups but it did not differ significantly from the control. Pups demonstrated normal growth rate as the body weight at birth and PND 4 and were comparable in treated and control groups. The body weight gain of pups at PND 0-4 were comparable in both control and treated groups. Gross observation of pups revealed no abnormalities attributable to the test chemical administration. Further, terminally sacrifice pups of all treated groups did not reveal lesion of pathological significance at any dose when compared with control. Pups died during the course of the study did not revealed treatment related extremal or visceral lesions. In summary, parental animals gained significantly less weight when male and female rats were orally administered with 1000 mg/kg of test chemical for 47-54 days. There was a marked reduction of fertility/pregnancy index at 1000 mg/kg but no significant alterations of other reproductive parameters were noted, therefore there productive NOAEL was1000mg/kg body weight/day. In addition, the prenatal and early postnatal exposure to the test chemical did not evoke adverse effect on neonatal survival, growth and development, when female Wistar rats were orally administered up to 1000 mg/kg for 54 days. Hence, the developmental NOAEL for F1 generation was 1000 mg/kg body weight/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

2. TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation.
- Weight at study initiation: Male: Minimum: 240 g Maximum: 315 g
Female: Minimum: 210 g Maximum: 260 g
- Fasting period before study: No data
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20[cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females. Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet from approved supplier was offered ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.30 to 22.70 °C
- Humidity (%): 43.90 to 67.60%
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: To: November 16, 2015 to March 26, 2016

3. TEST ANIMALS
- Source: Keari Inc.
- Age at study initiation: Females: 10 to 15 weeks
Males: 12 to 17 weeks.
- Weight at study initiation: Not documented
- Fasting period before study: Not documented
- Housing: Not documented
- Diet (e.g. ad libitum): Solid food purchased form Japan Kurea
- Water (e.g. ad libitum): Tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES: From: To: Not documented

Route of administration:

oral: gavage

Vehicle:

other: 2. corn oil; 3. olive oil

Details on exposure:

2. PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil. The test chemical was soluble in corn oil
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0.5 ml/100g body weight
- Lot/batch no. (if required): MR301015, MR161215
- Purity: No data

3. PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with olive oil to achieve concentration levels of 1000, 500, 100, 10 and 0 mg/ml

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil
- Concentration in vehicle: Not documented
- Amount of vehicle (if gavage): Not documented
- Lot/batch no. (if required): VDR7446
- Purity: Not documented

Details on mating procedure:

2.- M/F ratio per cage: One male and one female (1:1)
- Length of cohabitation: Female rats were housed with same male until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data
- Further matings after two unsuccessful attempts: Yes, Re-mating of unsuccessfully paired female was done with proven male of the same group.
- After successful mating each pregnant female was caged (how): No data
- Any other deviations from standard protocol: No data

3. On confirmation of the oestrous cycle of the females, 12-17 week old male rats were introduced and the males and females were co-housed from 5pm until the next morning when the presence of sperm in the vagina was considered to be successful mating. This was considered to be day zero of pregnancy. Based on their weight, pregnant rats were separated into 6 groups and relocated to separate cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated with respect to the following parameters.
Specificity:
The specificity will be evaluated by analysing the solvent used, standard solution, and sample solution.
Linearity:
The linearity was carried out by preparing and analyzing the standard solutions of at least 6 concentrations (covering the target analyte concentration i.e. 5 ppm,10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm ). A plot was drawn between the concentration and the response. The correlation coefficient, slope and intercept was calculated.
Assay accuracy and precision:
Assay accuracy and precision was carried out by fortifying the standard in vehicle at two levels (covering the target analyte concentration i.e., 10 ppm & 100 ppm). Five preparations were carried out at each concentration level selected. Two controls along with the assay accuracy samples were analysed. The mean, SD, % RSD was calculated. Assay accuracy was reported as the mean % recovery whereas the precision was reported as % RSD.
Homogeneity:
The homogeneity of the dose formulation prepared was determined by sampling and analyzing the formulation at top, middle and bottom layers. Sampling was done in two replicates from each layer.
Stability:
The stability of the prepared dose formulation was determined by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.

Duration of treatment / exposure:

2. 64 days
3. 10 days

Frequency of treatment:

2. Total days: 64
All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were further dosed till 47th day . Females were dosed during pregnancy and upto day 4 post partum.

3. Once daily from days 6 to 15 of pregnancy

Details on study schedule:

No data

Remarks:

Study 2.
0.0, 308.0, 556.0, 1000.0 mg/kg bw/day.

Study 3:
Doses / Concentrations: 0, 10, 100, 500, 1000 mg/kg bw/day
Basis: no data

No. of animals per sex per dose:

Study 2.
Total: 124 (104 Test animals + 20 recovery animals)
Test animals:
0 mg/Kg bw: 13 males and 13 females
308 mg/Kg bw: 13 males and 13 females
556 mg/Kg bw: 13 males and 13 females
1000 mg/Kg bw: 13 males and 13 females

Recovery animals:
0 mg/Kg bw: 5 males and 5 females
1000 mg/Kg bw: 5 males and 5 females

Study 3. 22 female rats in 1000mg/kg/day treatment group, 21 female rats in the 100 and 10 mg/kg/day treatment group and 20 female rats in the 500, 0 and non-administered treatment groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using validated software or the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights will be considered within ± 20% of the groups mean.
- Other: No data

Positive control:

No data

Parental animals: Observations and examinations:

2.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the acclimatization and study period
- Cage side observations checked in table [No.?] were included. Mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of animals of all groups were made once a day. Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Observations included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic
or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, During pre-mating, pregnancy and lactation, feed consumption were measured at least weekly. Feed consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT), Activated Partial Thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb) , Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN) – Calculated, Globulin (Glob) - Calculated, Alb/ Glb (A:G) – Calculated, Bile acids

URINALYSIS: Not specified
- Time schedule for collection of urine: Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION:Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

OTHER:
Functional Battery Observations: Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for five males and five females from control and treatment groups, during the last week of treatment and that of recovery groups, in the last week of recovery period.

Animals were subjected to examinations of various functional parameters which included; motor activity measurements using OPTO–VARIMEX 4, an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter; hind limb foot splay record and sensory reactivity measurements.

All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded. A more detailed weekly examination was performed throughout the treatment period. All animals found dead or killed for reasons of animal welfare were subjected to a thorough macroscopic examination of the visceral organs and specimens of abnormal tissues were retained. Males were weighed on the day that treatment commenced (F0) or the formal start of the generation (Fl), then weekly thereafter. F0 and Fl females were weighed on the same schedule until mating was detected and then on Days 0, 6, 13 and 20 after mating and on Days 1, 4, 7, 14 and 21 of lactation. Food consumption was recorded on a cage basis for F0 and Fl males and females weekly before pairing. Food consumption for females after mating was recorded daily on an individual basis on Days 0-5, 6-12 and 13-19 after mating. Food consumption for F0 females should have been recorded on Days 1-3, 4-6, 7-l3, and 14-20 of lactation but was recorded on Days 1-6, 7-13, 14-17 and 18-20 in error. Food consumption for Fl females was correctly recorded on Days 1-3, 4-6, 7-13, and 14-20 of lactation. After Day 14 of lactation, food intake is increasingly influenced by the offspring and is no longer an accurate
reflection of maternal intake.

3. Parent examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: Every 2 days
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, examined every 2 days.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
POST-MORTEM EXAMINATIONS: Yes - Sacrifice on gestation day 20
- Organs examined: No Data Available
OTHER: The implantation in the womb, corpus lutea quantity, the implantation quantity, the resorption embryo count and the living or dead foetuses. The weight of the placenta was measured.
Ovaries and uterine content:The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes - Number of early resorptions: No data - Number of late resorptions: No data - Other:
Fetal examinations
- External examinations: Yes: all living foetuses - Soft tissue examinations: Yes: half per litter - Skeletal examinations: Yes: - Head examinations: No data
Statistics
One way layout dispersion method if equal dispersion detected. The Kruskal-Wallis method was used to verify significance in the case of equal dispersion. The multi-comparison verification method of Scheffe and Dunnett was used to verify the significance of subjected groups. An X2 verification method was also performed to determine the frequency of of the bone changes, internal organs of the foetuses and the gender comparison of the surviving foetuses.

Oestrous cyclicity (parental animals):

2. Estrous cycle was monitored for its regularity during treatment period and in cohabitation for confirmation of pregnancy.

Sperm parameters (parental animals):

No Data Available

Litter observations:

2. STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No data
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. No data

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum. Live pups were weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No data

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No data

3. At term (on the 20th day of pregnancy) the fetuses were killed and examined to ascertaine the intrauterine death, external, internal and skeletal malformations.

Postmortem examinations (parental animals):

2. GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.

Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.

Following organs from randomly selected 5 male and 5 female rats were collected and preserved : Adrenals, Aorta, Bone (femur) with joint, Brain (cerebrum, cerebellum, mid brain), Cecum, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mammary glands, Mesenteric and Mandibular lymph node, Oesophagus, Ovaries with oviduct, Pancreas, Peyer's Patches, Pituitary, Prostate and Seminal vesicle with coagulating glands, Rectum, Salivary glands, Sciatic Nerve, Skeletal muscle, Skin, Spleen, Spinal Cord (cervical, mid-thoracic and lumbar), Sternum with marrow, Stomach, Testes, Thymus, Thyroid with Parathyroids, Trachea, Urinary Bladder, Uterus, Cervix with Vagina

Testes, epididymides, prostate and seminal vesicle with coagulating glands of all male rats and ovaries, uterus and cervix with vagina of all female rats were collected and preserved. Thyroid gland of one male and one female pup from each litter was collected and preserved. Collected Organs/tissues were fixed and preserved in 10 % Neutral buffered formalin. Testes and epididymis were initially fixed in Bouin’s fluid for approximately 24 hr, then 4 changes were given in 70% alcohol and transferred to 10 % neutral buffered formalin for preservation. Eyes were initially fixed in Modified Davidson‘s fluid for approximately 24 hr and then transferred to 10 % neutral buffered formalin for preservation.

Organ Weight: Weighing of brain, adrenals, ovaries with oviduct, testes, epididymides, heart, liver, kidneys, thymus and spleen was performed for randomly selected 5 male and 5 female rats. Testes and epididymides of all male rats were weighed. Before weighing adherent tissue/fat from organs were trimmed off and were kept in normal saline.

HISTOPATHOLOGY: Yes, All the preserved organs (Testes, epididymides, prostate and seminal vesicle with coagulating glands, ovaries, uterus and cervix with vagina) of all the rats, all the preserved tissues of randomly selected five male and five female rats of groups G1 and G4 and preserved thyroid of one male and one female pup of each litter were subjected to histopathological examination. All the tissues were trimmed, processed, embedded in paraffin wax. Sections were cut at a thickness of 3-5 micron and stained with hematoxylin and eosin stain. Processed tissues were subjected to histopathological examination. The prepared slides were examined under microscope by the Pathologist to note histopathological lesions, if any in different organs. Special attention was paid to observe effect of test item on reproductive system and spermatogenesis. The observed abnormalities were described according to morphological character, distribution, severity.

Postmortem examinations (offspring):

2. SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age. No data
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Gross pathology- Pups: Terminally sacrificed pups and pups died during the course of study were subjected to gross pathology

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. No data

3. At term (on the 20th day of pregnancy) the fetuses were killed and examined to ascertaine the intrauterine death, external, internal and skeletal malformations.

Statistics:

2. Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks

Reproductive indices:

2. Pregnancy index/fertility index ,Mean Post-Implantation Loss (%), Post-natal Loss (%) were determined

Offspring viability indices:

2. Pups Survival Index (%) was determined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

2.No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight). The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.

3. No significant changes in maternal parameters were observed in the all groups.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2. No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.

3. No significant changes in maternal parameters were observed in the all groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

2. A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight). Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.These changes observed were inconsistent, hence not considered as effect of the test item administration.

3. A slightly decreasing in maternal body weight gain was noted in the 1,000 mg/kg group, but not significantly.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

2.Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.

3. Food consumption during pregnancy in the test chemical-treated groups did not differ from the control group.

Food efficiency:

no effects observed

Description (incidence and severity):

2. Formulations were found to be homogeneous and stable upto 6 hour in vehicle corn oil. The mean active ingredient content at 61.6, 111.2 and 200 mg/ml concentration of the test chemical was 61.770, 110.321 and 200.007 mg/ml on day 1; 62.045, 110.902 and 198.199 mg/ml on day 21 and 60.726, 111.912 and 201.231 mg/ml on day 40, respectively

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed
for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R. The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistical ly significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.The above changes were inconsistent, not dose dependent hence considered as incidental in nature.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

2. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to therepective control group G1-R. The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration.
Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-R in female as compared to G1-R.The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2. Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5); Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5); Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5); Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5); Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5); Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5); Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild
cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5); Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5); Adrenal s: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5); Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13); Seminal Vesicles: multifocal mild neutrophilic /lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13); Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13). Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.
From the patho-morphological results presented, it is concluded that, the treatment of the test chemical at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs.

3. no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the Test Item.

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

2. No test item related changes in estrous cyclicity and precoital interval were observed. In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G3, 3 females and in group G4, 4 females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. Precoital Interval was calculated, all females showed precoital interval less than 5 days, except 1, 1 and 4 females from G1, G3 and G4, respectively which showed precoital interval more than 5 days.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

2. Pregnancy index was found to be 92.31, 84.62, 84.62 and 61.54 in G1, G2, G3 and G4 respectively. Marked decrease in Pregnancy index / Fertility index in G4(1000 mg/kg body weight) was considered to be treatment related.

3. No significant changes in maternal parameters were observed in the all groups.

Dose descriptor:

NOAEL

Effect level:

556 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Ophthalmological findings:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

2. There was no statistically significant difference between the control (G1) and treatment groups for mortality, no. of live births and survival index.
3. Intrauterine deaths did not show any significant increases in the fetuses treated by the test chemicals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. There was no statistically significant difference between the control (G1) and treatment groups for pups weight at birth and PND4 and weight gain at PND 4.
3. In both sexes of the 1,000 mg/kg group, body weight of the fetuses was significantly decreased.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. Pups died during course of study revealed various lesions among the control and treated groups viz., external examination emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54); Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54); Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and internal examination absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18); blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18); reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18); reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18); paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18); congested intestine (Female: G1: 1/56, G3: 1/54); autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18)

3. External malformations did not show any significant increases in the fetuses treated by BA. In the fetuses of 1,000 mg/kg group, internal and skeletal variations, such as dilation of the renal pelvis, wavy ribs, dumbbell shape of thoracic vertebra body, absence and splitting of thoracic vertebra body and lumber ribs, increased significantly.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

2. Pups sex ratio (Male/Female) was found to be 55/57, 44/30, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

556 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

other: Fertility index/pregnancy index was decreased at dose level of 1000 mg/Kg bw in dams which was correlated to the viability of offsprings

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

The reproductive and developmental NOAEL was 1000 mg/kg body weight/day based on the absence of adverse effect on fertility and reproductive performance and offspring development when male and female Wistar rats were orally administered the test chemical up to 1000 mg/kg for 47-54 days.

Executive summary:

Reproductive Toxicity Study:

 

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical. The reviewed studies are the follows:

 

Study 2:

A reproduction/developmental toxicity screening test (OECD TG 422) was conducted on male and female Wistar rats to assess thereproductive performance and offspring developmentfollowing oral administration ofthe test chemical. Groups of 13 rats/sex/dose were orally administered with 0 (vehicle control, corn oil), 308, 556 and 1000 mg/kg of test chemical.Groups of 5 rats/sex were administered either 0 or 1000 mg/kg test chemical daily till the first scheduled sacrifice and kept for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. All animals of both sexes were dosed 2 weeks prior to mating and during the mating period. Males were further dosed till day 47th day. Female rats were dosed during pregnancy till 4 day of postpartum.The oestrus cycle was monitored daily from the beginning of treatment until the evidence of mating. All animals were observed for mortality and morbidity twice daily. General clinical observations of animals in all groups were made daily, detailed clinical examination was performed once before the first treatment, then weekly. Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for 5 males and 5 females of each groups during the last week of treatment and for recovery groups during the last recovery week. The body weight and feed consumption were weighted weekly for both males and females. Pregnant females were weighted on day 0, 7, 14 and 20, post-partum day 0-1, on day 4 post-partum and before sacrifice. Haematology and clinical biochemistry were performed on 5 males and 5 females of each group, prior to necropsy and organs weights measurements. On termination day, all animals were subjected to gross pathological examination, ovaries, uterus, cervix with vagina, testes, epididymis, prostate, seminal vesicle with coagulation gland were preserved. Full histopathology of the preserved sexual organs of all animals and all tissues of five males and five females randomly selected from control and high-dose group. No mortality and morbidity were observed in both treatment and recovery groups during the entire study period. No apparent treatment-related clinical sign was observed in the treatment and recovery groups. Sensory reactivity and motor activity measurements were comparable, and no treatment related changes were revealed at any dose level when compared to control groups.No treatment-related and dose-dependent changes in mean body weight were noted up to 1000 mg/kg both in treatment and recovery groups. However, the mean body weight change (%) of males decreased significantly during the entire treatment period (Day 1-46) at 1000 mg/kg and during the first two weeks of treatment at 556 mg/kg. Males from the recovery group also demonstrated significant decrease in the mean body weight changes during day 1-29.Females gained significantly less weight during gestation (days 1-20), but the mean body weight change did not alter in the recovery group.The feed consumption of all treatment and recovery groups of both sexes were comparable and did not show any significant differences as compared with respective controls. In addition, no treatment related and/or dose dependent changes were noted in haematological and clinical biochemistry parameters up to 1000 mg/kg body weight. External and visceral examination of all treated and control rats including recovery groups did not reveal any abnormality of pathological significance. The absolute and relative organ weights of brain, adrenals, ovaries with oviduct, testes, epididymis, heart, liver, kidney spleen, thymus of dosed rats of either sex did not differ significantly when compared to respective control groups. Oestrus cyclicity was regular i.e. 3-5 days in control, treatment and recovery groups. During cohabitation, all females from treatment and control groups showed evidence of copulation. Most females showed pre-coital interval less than 5 days, except 4 females from the 1000 mg/kg, 1-1 females from 556 mg/kg and control groups which showed pre-coital interval longer than 5 days. Only 8 out of 13 females achieved pregnancy in the high-dose group and the pregnancy/fertility index decreased to 61.54%. The gestation length was comparable in all treatment and control groups. Examination of the uterus content revealed that the number of implantation and corpora lutea did not differ in treated and control rats. The reproductive organs of all dosed rats were subjected to histopathological evaluation. Histopathological lesions observed in males included focal minimal to mild seminiferous tubule degeneration, focal minimal to mild and multifocal minimal retention of mature sperm, focal minimal to mild sloughing of round spermatid/pachytene spermatocyte. Histopathological lesions observed in females included minimal reduction of stromal cells, focal moderate necrosis and nodular hyperplasia of uterus as well as minimal lymphocytic infiltration of the cervix. However, the severity and incidence of histopathological lesions of testis, uterus and cervix were well comparable with respective control groups and exhibited no dose relationship. Hence, the occurrence of these lesions was considered spontaneous or incidental in nature and did not attributed to the test chemical administration. Developmental parameters such as number of live births, litter size, number of alive pups at PND 4, and post-implantation loss were comparable in all groups. Postnatal loss (%)wereincreased at 556 and 1000 mg/kg but was not statistically significant. The fetal survival index at PND 4 slightly decreased in middle and top dose groups but it did not differ significantly from the control. Pups demonstrated normal growth rate as the body weight at birth and PND 4 and were comparable in treated and control groups. The body weight gain of pups at PND 0-4 were comparable in both control and treated groups. Gross observation of pups revealed no abnormalities attributable to the test chemical administration. Further, terminally sacrifice pups of all treated groups did not reveal lesion of pathological significance at any dose when compared with control.Pups died during the course of the study did not revealed treatment related extremal or visceral lesions.In summary,parental animals gained significantly less weight when male and female rats were orally administered with 1000 mg/kg of test chemical for 47-54 days. There was a marked reduction of fertility/pregnancy index at 1000 mg/kg but no significant alterations of other reproductive parameters were noted, therefore thereproductive NOAEL was1000mg/kg body weight/day.In addition, the prenatal and early postnatal exposure to the test chemical did not evoke adverse effect on neonatal survival, growth and development, when female Wistar rats were orally administered up to 1000 mg/kg for 54 days. Hence, the developmental NOAEL for F1 generation was 1000 mg/kg body weight/day.

 

Study 3:

A prenatal developmental toxicity study was performed to investigate the toxic effect of the test chemical on foetus organogenesis and development. Groups of 20 pregnant Wistar rats were administered 0, 10, 100, 500, or 1,000 mg/kg bw/day by gavage during gestation days 6-15 (GD 6-15). On day 20 of gestation, pregnancies were terminated and the fetuses were examined for intrauterine death, and internal, external and skeletal malformations. Maternal parameters included mortality, body weight, food consumption, and clinical and gross examinations. No maternal toxic effects on parameters examined were observed as no death and alterations of body weight, food consumption was reported, and clinical and gross examinations did not reveal any effect attributable to test chemical administration. Examination of the uterus content revealed that the number of implantation and corpora lutea, live/dead fetuses, or resorptions, implantation ratio, sex ratio, or placenta weigh did not differ in treated and control rats.The fetal body weight was significantly decreased at 1000 mg/kg and significantly increased at low and middle doses, thus these alterations were inconclusive and showed no dose-dependence.There was a statistically significant increase in the combined incidence of organ variations (i.e., slight dilatation of the lateral ventricle and renal pelvis, and presence of levo-umbilical artery) in animals from the 500 and 1000 mg/kg dose groups. The only skeletal malformation (fused ribs) was seen in one fetus of the high-dose group, which did not increase the incidence of skeletal malformations compared to controls. The authors suggested that the skeletal malformations were related to the significant decrease in fetal body weight. Skeletal variations (i.e., wavy ribs, dumbbell shaped vertebrae, absence/splitting of thoracic vertebrae, presence of lumbar ribs and degree of ossification) were statistically increased at 1000 mg/kg. No increase in intrauterine death or external variations was noted at any dose level. In conclusion, the oral administration of the test chemical up to 1000 mg/kg was not maternally toxic evidenced by the absence of alterations of parameters examined. In addition, the prenatal exposure to 1000 mg/kg of the test chemical did not alter the normal growth and development of offspring and, consequently the developmental NOAEL was 1000 mg/kg for the F1 generation.   

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The study is ongoing and this information will be submitted later based on ECHA communication/decision number CCH-D-2114536388-40-01/F.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive Toxicity Study:

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical.The studies are the follows.

Study 2:

A reproduction/developmental toxicity screening test (OECD TG 422) was conducted on male and female Wistar rats to assess the reproductive performance and offspring development following oral administration of the test chemical. Groups of 13 rats/sex/dose were orally administered with 0 (vehicle control, corn oil), 308, 556 and 1000 mg/kg of test chemical. Groups of 5 rats/sex were administered either 0 or 1000 mg/kg test chemical daily till the first scheduled sacrifice and kept for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. All animals of both sexes were dosed 2 weeks prior to mating and during the mating period. Males were further dosed till day 47th day. Female rats were dosed during pregnancy till 4 day of postpartum. The oestrus cycle was monitored daily from the beginning of treatment until the evidence of mating. All animals were observed for mortality and morbidity twice daily. General clinical observations of animals in all groups were made daily, detailed clinical examination was performed once before the first treatment, then weekly. Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for 5 males and 5 females of each groups during the last week of treatment and for recovery groups during the last recovery week. The body weight and feed consumption were weighted weekly for both males and females. Pregnant females were weighted on day 0, 7, 14 and 20, post-partum day 0-1, on day 4 post-partum and before sacrifice. Haematology and clinical biochemistry were performed on 5 males and 5 females of each group, prior to necropsy and organs weights measurements. On termination day, all animals were subjected to gross pathological examination, ovaries, uterus, cervix with vagina, testes, epididymis, prostate, seminal vesicle with coagulation gland were preserved. Full histopathology of the preserved sexual organs of all animals and all tissues of five males and five females randomly selected from control and high-dose group. No mortality and morbidity were observed in both treatment and recovery groups during the entire study period. No apparent treatment-related clinical sign was observed in the treatment and recovery groups. Sensory reactivity and motor activity measurements were comparable, and no treatment related changes were revealed at any dose level when compared to control groups. No treatment-related and dose-dependent changes in mean body weight were noted up to 1000 mg/kg both in treatment and recovery groups. However, the mean body weight change (%) of males decreased significantly during the entire treatment period (Day 1-46) at 1000 mg/kg and during the first two weeks of treatment at 556 mg/kg. Males from the recovery group also demonstrated significant decrease in the mean body weight changes during day 1-29. Females gained significantly less weight during gestation (days 1-20), but the mean body weight change did not alter in the recovery group. The feed consumption of all treatment and recovery groups of both sexes were comparable and did not show any significant differences as compared with respective controls. In addition, no treatment related and/or dose dependent changes were noted in haematological and clinical biochemistry parameters up to 1000 mg/kg body weight. External and visceral examination of all treated and control rats including recovery groups did not reveal any abnormality of pathological significance. The absolute and relative organ weights of brain, adrenals, ovaries with oviduct, testes, epididymis, heart, liver, kidney spleen, thymus of dosed rats of either sex did not differ significantly when compared to respective control groups. Oestrus cyclicity was regular i.e. 3-5 days in control, treatment and recovery groups. During cohabitation, all females from treatment and control groups showed evidence of copulation. Most females showed pre-coital interval less than 5 days, except 4 females from the 1000 mg/kg, 1-1 females from 556 mg/kg and control groups which showed pre-coital interval longer than 5 days. Only 8 out of 13 females achieved pregnancy in the high-dose group and the pregnancy/fertility index decreased to 61.54%. The gestation length was comparable in all treatment and control groups. Examination of the uterus content revealed that the number of implantation and corpora lutea did not differ in treated and control rats. The reproductive organs of all dosed rats were subjected to histopathological evaluation. Histopathological lesions observed in males included focal minimal to mild seminiferous tubule degeneration, focal minimal to mild and multifocal minimal retention of mature sperm, focal minimal to mild sloughing of round spermatid/pachytene spermatocyte. Histopathological lesions observed in females included minimal reduction of stromal cells, focal moderate necrosis and nodular hyperplasia of uterus as well as minimal lymphocytic infiltration of the cervix. However, the severity and incidence of histopathological lesions of testis, uterus and cervix were well comparable with respective control groups and exhibited no dose relationship. Hence, the occurrence of these lesions was considered spontaneous or incidental in nature and did not attributed to the test chemical administration. Developmental parameters such as number of live births, litter size, number of alive pups at PND 4, and post-implantation loss were comparable in all groups. Postnatal loss (%) were increased at 556 and 1000 mg/kg but was not statistically significant. The fetal survival index at PND 4 slightly decreased in middle and top dose groups but it did not differ significantly from the control. Pups demonstrated normal growth rate as the body weight at birth and PND 4 and were comparable in treated and control groups. The body weight gain of pups at PND 0-4 were comparable in both control and treated groups. Gross observation of pups revealed no abnormalities attributable to the test chemical administration. Further, terminally sacrifice pups of all treated groups did not reveal lesion of pathological significance at any dose when compared with control. Pups died during the course of the study did not revealed treatment related extremal or visceral lesions. In summary, parental animals gained significantly less weight when male and female rats were orally administered with 1000 mg/kg of test chemical for 47-54 days. There was a marked reduction of fertility/pregnancy index at 1000 mg/kg but no significant alterations of other reproductive parameters were noted, therefore there productive NOAEL was1000mg/kg body weight/day. In addition, the prenatal and early postnatal exposure to the test chemical did not evoke adverse effect on neonatal survival, growth and development, when female Wistar rats were orally administered up to 1000 mg/kg for 54 days. Hence, the developmental NOAEL for F1 generation was 1000 mg/kg body weight/day.

Study 3:

A prenatal developmental toxicity study was performed to investigate the toxic effect of the test chemical on foetus organogenesis and development. Groups of 20 pregnant Wistar rats were administered 0, 10, 100, 500, or 1,000 mg/kg bw/day by gavage during gestation days 6-15 (GD 6-15). On day 20 of gestation, pregnancies were terminated and the fetuses were examined for intrauterine death, and internal, external and skeletal malformations. Maternal parameters included mortality, body weight, food consumption, and clinical and gross examinations. No maternal toxic effects on parameters examined were observed as no death and alterations of body weight, food consumption was reported, and clinical and gross examinations did not reveal any effect attributable to test chemical administration. Examination of the uterus content revealed that the number of implantation and corpora lutea, live/dead fetuses, or resorptions, implantation ratio, sex ratio, or placenta weigh did not differ in treated and control rats. The fetal body weight was significantly decreased at 1000 mg/kg and significantly increased at low and middle doses, thus these alterations were inconclusive and showed no dose-dependence. There was a statistically significant increase in the combined incidence of organ variations (i.e., slight dilatation of the lateral ventricle and renal pelvis, and presence of levo-umbilical artery) in animals from the 500 and 1000 mg/kg dose groups. The only skeletal malformation (fused ribs) was seen in one fetus of the high-dose group, which did not increase the incidence of skeletal malformations compared to controls. The authors suggested that the skeletal malformations were related to the significant decrease in fetal body weight. Skeletal variations (i.e., wavy ribs, dumbbell shaped vertebrae, absence/splitting of thoracic vertebrae, presence of lumbar ribs and degree of ossification) were statistically increased at 1000 mg/kg. No increase in intrauterine death or external variations was noted at any dose level. In conclusion, the oral administration of the test chemical up to 1000 mg/kg was not maternally toxic evidenced by the absence of alterations of parameters examined. In addition, the prenatal exposure to 1000 mg/kg of the test chemical did not alter the normal growth and development of offspring and, consequently the developmental NOAEL was 1000 mg/kg for the F1 generation.   

Effects on developmental toxicity

Description of key information

Developmental toxicity study

The possible adverse effect ofthe test chemicalon foetus/prenatal development was tested using mated female Sprague-Dawley rats. The test chemical was dissolved in corn oil and administered to 24 rats/dose on the dosage levels of 0 (corn oil), 50, 200, 500 mg/kg/day via oral gavage during gestation days 6-15. Animals were observed for mortality and clinical signs, changes in body weight and food consumption were recorded as well. In utero parameters such as the numbers of total implantations, post-implantation loss, fetal resorption were recorded as well as the litter weight and fetal viability. Approximately 1/2 of the fetuses in each litter were processed for soft-tissue evaluations while the other half for skeletal evaluations.Twodeaths occurred at 500 mg/kg. Excessive alopecia, salivation and/or anogenital staining was observedbut no pattern of treatment relationship could be determined.Statistically reduced maternal weightgain and food consumption were observed at 200 and 500 mg/kg.The prenatal exposure to the test chemical did not have effecton fetal resorptions, post-implantation loss fetal viability, ortotalnumber ofimplantations.The mean litter weights in treated and control groupswerecomparable. No significant increases were observed in incidence ofmalformations or variations at any treatment level.In conclusion, the oral administration ofthe test chemical to pregnant SD ratswas maternally toxic evidenced by the treatment related clinical signs andreduced maternal weightgain and food consumption at 200 and 500 mg/kg. Hence, the NOAEL for systemic toxicity was 50 mg/kg/day. No changes in maternal developmental parameters were observed at any dose level when pregnant SD rats were orally dosed during GD 6-15 and therefore the maternal developmental NOAEL was 500 mg/kg body weight/day. In addition, the prenatal exposure to the test chemical up to 500 mg/kg had no effect on growth and survival of the foetuses, and/or on the incidences in external, skeletal and soft tissue malformations and variations in fetuses and, therefore the developmental NOAEL for was 500 mg/kg body weight/day for the F1 generation.    

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on developmental toxicity studies on rats

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 1.Wistar 2.Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation.
- Weight at study initiation: Male: Minimum: 240 g Maximum: 315 g
Female: Minimum: 210 g Maximum: 260 g
- Fasting period before study: No data
- Housing: A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20[cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females. Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet was offered ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: A conventional laboratory pelleted diet was offered. Aqua guard filtered drinking water in bottles was offered.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.30 to 22.70 °C
- Humidity (%): 43.90 to 67.60%
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: To: November 16, 2015 to March 26, 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

1.PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil. The test chemical was soluble in corn oil
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0.5 ml/100g body weight
- Lot/batch no. (if required): MR301015, MR161215
- Purity: No data
2.PREPARATION OF DOSING SOLUTIONS:
Test material 73.5% & 26.5% biphenyl mixed in corn oil.
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 50, 200, 500 mg/kg/day - Amount of vehicle (if gavage): 5ml/kg

- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated with respect to the following parameters.

Specificity:
The specificity will be evaluated by analysing the solvent used, standard solution, and sample solution.

Linearity:
The linearity was carried out by preparing and analyzing the standard solutions of at least 6 concentrations (covering the target analyte concentration i.e. 5 ppm,10 ppm, 25 ppm, 50 ppm, 75 ppm and 100 ppm ). A plot was drawn between the concentration and the response. The correlation coefficient, slope and intercept was calculated.

Assay accuracy and precision:
Assay accuracy and precision was carried out by fortifying the standard in vehicle at two levels (covering the target analyte concentration i.e., 10 ppm & 100 ppm). Five preparations were carried out at each concentration level selected. Two controls along with the assay accuracy samples were analysed. The mean, SD, % RSD was calculated. Assay accuracy was reported as the mean % recovery whereas the precision was reported as % RSD.

Homogeneity:
The homogeneity of the dose formulation prepared was determined by sampling and analyzing the formulation at top, middle and bottom layers. Sampling was done in two replicates from each layer.

Stability:
The stability of the prepared dose formulation was determined by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.

Details on mating procedure:

- M/F ratio per cage: One male and one female (1:1)
- Length of cohabitation: Female rats were housed with same male until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Yes, Re-mating of unsuccessfully paired female was done with proven male of the same group.
- After successful mating each pregnant female was caged (how): No data
- Any other deviations from standard protocol: No data

Duration of treatment / exposure:

1.Total days: 64
All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were further dosed till 47th day . Females were dosed during pregnancy and upto day 4 post partum.
2.10 days ( on gestation days 6-15)Details on exposure

Frequency of treatment:

Daily

Duration of test:

Study1.64 days
Study2.15 days

Remarks:

Study1.
0.0,308.0,556.0,1000mg/kg/day
Study2.
0,50, 200, 500 mg/kg/day

No. of animals per sex per dose:

Study1.Total: 124 ( 104 Test animals + 20 recovery animals)
Test animals:
0 mg/Kg bw: 13 males and 13 females
308 mg/Kg bw: 13 males and 13 females
556 mg/Kg bw: 13 males and 13 females
1000 mg/Kg bw: 13 males and 13 females

Recovery animals:
0 mg/Kg bw: 5 males and 5 females
1000 mg/Kg bw: 5 males and 5 females
Study2.Total:96
0 mg/kg bw/day:24
50mg/kg bw/day:24
200mg/kg bw/day:24
500mg/kg bw/day:24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using validated software or the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights will be considered within ± 20% of the groups mean.
- Other: No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the acclimatization and study period
- Cage side observations checked in table [No.?] were included. Mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of animals of all groups were made once a day. Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

Observations included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, During pre-mating, pregnancy and lactation, feed consumption were measured at least weekly. Feed consumption was not measured during mating period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT), Activated Partial Thromboplastin time (aPTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to necropsy at the end of the treatment and recovery periods
- Animals fasted: Yes, Animals were fasted overnight (approximately 16-18 hr) prior to blood collection
- How many animals: 5 males and 5 females
- Parameters checked in table [No.?] were examined. Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb) , Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN) – Calculated, Globulin (Glob) - Calculated, Alb/ Glb (A:G) – Calculated, Bile acids

URINALYSIS: Not specified
- Time schedule for collection of urine: Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION:Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

OTHER:
Functional Battery Observations: Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for five males and five females from control and treatment groups, during the last week of treatment and that of recovery groups, in the last week of recovery period.

Animals were subjected to examinations of various functional parameters which included; motor activity measurements using OPTO–VARIMEX 4, an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter; hind limb foot splay record and sensory reactivity measurements.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:

Fetal examinations:

1- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes:

2.- External examinations: Yes:
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Head examinations: No data

Statistics:

1.Raw data was analysed using statistical software “Sigma Plot 11.0”. The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks
2.Statistical evaluation of equality of means was made by the appropriate one-way analysis of variance technique (ANOVA) for parametric procedures and Kruskal-Wallis test for nonparametric procedures were used after applying Bartlett's test for determination of equal variance. Statistical tests for trend, using either standard regression techniques (parametric cases) or Jonckheere's test in nonparametric cases. Levels of statistical significance used were either p<0.05 or p<0.01.

Indices:

Pregnancy index/fertility index was determined

Historical control data:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1.No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.

Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight).

The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.
2.Excessive alopecia, salivation and/or anogenital staining was observed but no pattern of treatment relationship could be determined.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

1.No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.
2.2 deaths occurred at 500 mg/kg

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

1.A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight).

Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.

These changes observed were inconsistent, hence not considered as effect of the test item administration.
2.Statistically reduced maternal weight gain were observed at 200 and 500 mg/kg/d.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1.Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.
2.Statistically reduced food consumption were observed at 200 and 500 mg/kg/d.

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R.

The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistically significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.

The above changes were inconsistent, not dose dependent hence considered as incidental in nature.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.

Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to the respective control group G1-R.

The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration.

Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-R in female as compared to G1-R.

The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of treatment and recovery period, absolute and relative weight of organs of treated rats of either sex did not differ significantly except a significant increase in relative wieght of Adrenal of G4-R male group when compared to the respective control group rats.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

External Findings: External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance.

Internal Findings: Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes:

Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5);

Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5);

Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5);

Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5);

Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5);

Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5);

Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5);

Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5);

Adrenals: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5);

Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13);

Seminal Vesicles: multifocal mild neutrophilic /lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13);

Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13).

Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.

From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the Test Item.

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effects observed on postimplantation loss or total implantations

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy index was found to be 92.31, 84.62, 84.62 and 61.54 in G1, G2, G3 and G4 respectively. Marked decrease in Pregnancy index / Fertility index in G4(1000 mg/kg body weight) was considered to be treatment related.

Dose descriptor:

NOAEL

Effect level:

556 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: decrease in pregancy index was observed in 1000mg/kg bw dose group

Abnormalities:

not specified

Localisation:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

1.There was no statistically significant difference between the control (G1) and treatment groups for pups weight at birth and PND4 and weight gain at PND4.
2.Mean litter weights in treated and control groups were similar
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

Pups sex ratio (Male/Female) was found to be 55/57, 33/40, 43/58, and 21/26 in G1, G2, G3 and G4 respectively.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

1.Pups died during course of study revealed various lesions among the control and treated groups viz.,
External examination
Emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54);
Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54);
Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and

Internal examination:
Absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18);
Blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18);
Reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18);
Reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18);
Paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18);
Congested intestine (Female: G1: 1/56, G3: 1/54);
Autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18)
2.No increases were observed in incidence of malformations or variations at any treatment level.

Skeletal malformations:

no effects observed

Visceral malformations:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

556 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

Remarks on result:

other: No developmental toxic effects was observed

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

The developmental NOAEL is concluded as 500 mg/kg/day for both maternal and the F1 generations.

Executive summary:

Available data on read across chemicals from developmental toxicity studies was summarized to evaluate the developmental toxicity of test chemical. The prenatal developmental toxicity studies are the follows:

Study 2:

A reproduction/developmental toxicity screening test (OECD TG 422) was conducted on male and female Wistar rats to assess the reproductive performance and offspring development following oral administration of the test chemical. Groups of 13 rats/sex/dose were orally administered with 0 (vehicle control, corn oil), 308, 556 and 1000 mg/kg of test chemical. Groups of 5 rats/sex were administered either 0 or 1000 mg/kg test chemical daily till the first scheduled sacrifice and kept for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. All animals of both sexes were dosed 2 weeks prior to mating and during the mating period. Males were further dosed till day 47th day. Female rats were dosed during pregnancy till 4 day of postpartum. The oestrus cycle was monitored daily from the beginning of treatment until the evidence of mating. All animals were observed for mortality and morbidity twice daily. General clinical observations of animals in all groups were made daily, detailed clinical examination was performed once before the first treatment, then weekly. Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for 5 males and 5 females of each groups during the last week of treatment and for recovery groups during the last recovery week. The body weight and feed consumption were weighted weekly for both males and females. Pregnant females were weighted on day 0, 7, 14 and 20, post-partum day 0-1, on day 4 post-partum and before sacrifice. Haematology and clinical biochemistry were performed on 5 males and 5 females of each group, prior to necropsy and organs weights measurements. On termination day, all animals were subjected to gross pathological examination, ovaries, uterus, cervix with vagina, testes, epididymis, prostate, seminal vesicle with coagulation gland were preserved. Full histopathology of the preserved sexual organs of all animals and all tissues of five males and five females randomly selected from control and high-dose group. No mortality and morbidity were observed in both treatment and recovery groups during the entire study period. No apparent treatment-related clinical sign was observed in the treatment and recovery groups. Sensory reactivity and motor activity measurements were comparable, and no treatment related changes were revealed at any dose level when compared to control groups. No treatment-related and dose-dependent changes in mean body weight were noted up to 1000 mg/kg both in treatment and recovery groups. However, the mean body weight change (%) of males decreased significantly during the entire treatment period (Day 1-46) at 1000 mg/kg and during the first two weeks of treatment at 556 mg/kg. Males from the recovery group also demonstrated significant decrease in the mean body weight changes during day 1-29. Females gained significantly less weight during gestation (days 1-20), but the mean body weight change did not alter in the recovery group. The feed consumption of all treatment and recovery groups of both sexes were comparable and did not show any significant differences as compared with respective controls. In addition, no treatment related and/or dose dependent changes were noted in haematological and clinical biochemistry parameters up to 1000 mg/kg body weight. External and visceral examination of all treated and control rats including recovery groups did not reveal any abnormality of pathological significance. The absolute and relative organ weights of brain, adrenals, ovaries with oviduct, testes, epididymis, heart, liver, kidney spleen, thymus of dosed rats of either sex did not differ significantly when compared to respective control groups. Oestrus cyclicity was regular i.e. 3-5 days in control, treatment and recovery groups. During cohabitation, all females from treatment and control groups showed evidence of copulation. Most females showed pre-coital interval less than 5 days, except 4 females from the 1000 mg/kg, 1-1 females from 556 mg/kg and control groups which showed pre-coital interval longer than 5 days. Only 8 out of 13 females achieved pregnancy in the high-dose group and the pregnancy/fertility index decreased to 61.54%. The gestation length was comparable in all treatment and control groups. Examination of the uterus content revealed that the number of implantation and corpora lutea did not differ in treated and control rats. The reproductive organs of all dosed rats were subjected to histopathological evaluation. Histopathological lesions observed in males included focal minimal to mild seminiferous tubule degeneration, focal minimal to mild and multifocal minimal retention of mature sperm, focal minimal to mild sloughing of round spermatid/pachytene spermatocyte. Histopathological lesions observed in females included minimal reduction of stromal cells, focal moderate necrosis and nodular hyperplasia of uterus as well as minimal lymphocytic infiltration of the cervix. However, the severity and incidence of histopathological lesions of testis, uterus and cervix were well comparable with respective control groups and exhibited no dose relationship. Hence, the occurrence of these lesions was considered spontaneous or incidental in nature and did not attributed to the test chemical administration. Developmental parameters such as number of live births, litter size, number of alive pups at PND 4, and post-implantation loss were comparable in all groups. Postnatal loss (%) increased at 556 and 1000 mg/kg but it was not statistically significant. The fetal survival index at PND 4 slightly decreased in middle and top dose groups but it did not differ significantly from the control. Pups demonstrated normal growth rate as the body weight at birth and PND 4 and were comparable in treated and control groups. The body weight gain of pups at PND 0-4 were comparable in both control and treated groups. Gross observation of pups revealed no abnormalities attributable to the test chemical administration. Further, terminally sacrifice pups of all treated groups did not reveal any lesion of pathological significance at any dose when compared with control. Pups died during the course of the study did not revealed treatment related extremal or visceral lesions. In summary, parental animals gained significantly less weight when male and female rats were orally administered with 1000 mg/kg of test chemical for 47-54 days. In addition, the prenatal and early postnatal exposure to the test chemical did not evoke adverse effect on neonatal survival, growth and development, when female Wistar rats were orally administered up to 1000 mg/kg for 54 days. Hence, the developmental NOAEL for F1 generation was 1000 mg/kg body weight/day.Similarly, the maternal developmental NOAEL was 1000 mg/kg/day based on the lack of significant alterations of maternal developmental (in utero) parameters examined.

Study 3:

The possible adverse effect of the test chemical on foetus/prenatal development was tested using mated female Sprague-Dawley rats. The test chemical was dissolved in corn oil and administered to 24 rats/dose on the dosage levels of 0 (corn oil), 50, 200, 500 mg/kg/day via oral gavage during gestation days 6-15. Animals were observed for mortality and clinical signs, changes in body weight and food consumption were recorded as well. In utero parameters such as the numbers of total implantations, post-implantation loss, fetal resorption were recorded as well as the litter weight and fetal viability. Approximately 1/2 of the fetuses in each litter were processed for soft-tissue evaluations while the other half for skeletal evaluations. Two deaths occurred at 500 mg/kg. Excessive alopecia, salivation and/or anogenital staining was observed but no pattern of treatment relationship could be determined. Statistically reduced maternal weight gain and food consumption were observed at 200 and 500 mg/kg. The prenatal exposure to the test chemical did not have effect on fetal resorptions, post-implantation loss fetal viability, or total number of implantations. The mean litter weights in treated and control groups were comparable. No significant increases were observed in incidence of malformations or variations at any treatment level. In conclusion, the oral administration of the test chemical to pregnant SD rats was maternally toxic evidenced by the treatment related clinical signs and reduced maternal weight gain and food consumption at 200 and 500 mg/kg. Hence, the NOAEL for systemic toxicity was 50 mg/kg/day. No changes in maternal developmental parameters were observed at any dose level when pregnant SD rats were orally dosed during GD 6-15 and therefore the maternal developmental NOAEL was 500 mg/kg body weight/day. In addition, the prenatal exposure to the test chemical up to 500 mg/kg had no effect on growth and survival of the foetuses, and/or on the incidences in external, skeletal and soft tissue malformations and variations in fetuses and, therefore the developmental NOAEL for was 500 mg/kg body weight/day for the F1 generation.    

Study 4:

A prenatal developmental toxicity study was performed to investigate the toxic effect of the test chemical on fetus organogenesis and development. Groups of 20 pregnant Wistar rats were administered 0, 10, 100, 500, or 1,000 mg/kg bw/day by gavage during gestation days 6-15 (GD 6-15). On day 20 of gestation, pregnancies were terminated, and the fetuses were examined for intrauterine death, and internal, external and skeletal malformations. Maternal parameters included mortality, body weight, food consumption, and clinical and gross examinations. No maternal toxic effects on parameters examined were observed as no death and alterations of body weight, food consumption was reported, and clinical and gross examinations did not reveal any effect attributable to test chemical administration. Examination of the uterus content revealed that the number of implantation and corpora lutea, live/dead fetuses, or resorptions, implantation ratio, sex ratio, or placenta weigh did not differ in treated and control rats.The fetal body weight was significantly decreased at 1000 mg/kg and significantly increased at low and middle doses, thus these alterations were inconclusive and showed no dose-dependence.There was a statistically significant increase in the combined incidence of organ variations (i.e., slight dilatation of the lateral ventricle and renal pelvis, and presence of levo-umbilical artery) in animals from the 500 and 1000 mg/kg dose groups. The only skeletal malformation (fused ribs) was seen in one fetus of the high-dose group, which did not increase the incidence of skeletal malformations compared to controls. The authors suggested that the skeletal malformations were related to the significant decrease in fetal body weight. Skeletal variations (i.e., wavy ribs, dumbbell shaped vertebrae, absence/splitting of thoracic vertebrae, presence of lumbar ribs and degree of ossification) were statistically increased at 1000 mg/kg. However, skeletal variations are minor structural changes which have little or no detrimental effect on the animal and often are transient changes. No increase in intrauterine death or external variations was noted at any dose level.In conclusion, the oral administration of the test chemical up to 1000 mg/kg was not maternally toxic evidenced by the absence of alterations of parameters examined. In addition, the prenatal exposure to 1000 mg/kg of the test substance did not alter the normal growth and development of offspring and, consequently the developmental NOAEL was 1000 mg/kg for the F1 generation.   

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity study:

Data available from different studies were reviewed to determine the developmental toxicity of test chemical. The studies are the follows:

Study 2:

A reproduction/developmental toxicity screening test (OECD TG 422) was conducted on male and female Wistar rats to assess the reproductive performance and offspring development following oral administration of the test chemical. Groups of 13 rats/sex/dose were orally administered with 0 (vehicle control, corn oil), 308, 556 and 1000 mg/kg of test chemical. Groups of 5 rats/sex were administered either 0 or 1000 mg/kg test chemical daily till the first scheduled sacrifice and kept for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. All animals of both sexes were dosed 2 weeks prior to mating and during the mating period. Males were further dosed till day 47th day. Female rats were dosed during pregnancy till 4 day of postpartum. The oestrus cycle was monitored daily from the beginning of treatment until the evidence of mating. All animals were observed for mortality and morbidity twice daily. General clinical observations of animals in all groups were made daily, detailed clinical examination was performed once before the first treatment, then weekly. Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for 5 males and 5 females of each groups during the last week of treatment and for recovery groups during the last recovery week. The body weight and feed consumption were weighted weekly for both males and females. Pregnant females were weighted on day 0, 7, 14 and 20, post-partum day 0-1, on day 4 post-partum and before sacrifice. Haematology and clinical biochemistry were performed on 5 males and 5 females of each group, prior to necropsy and organs weights measurements. On termination day, all animals were subjected to gross pathological examination, ovaries, uterus, cervix with vagina, testes, epididymis, prostate, seminal vesicle with coagulation gland were preserved. Full histopathology of the preserved sexual organs of all animals and all tissues of five males and five females randomly selected from control and high-dose group. No mortality and morbidity were observed in both treatment and recovery groups during the entire study period. No apparent treatment-related clinical sign was observed in the treatment and recovery groups. Sensory reactivity and motor activity measurements were comparable, and no treatment related changes were revealed at any dose level when compared to control groups. No treatment-related and dose-dependent changes in mean body weight were noted up to 1000 mg/kg both in treatment and recovery groups. However, the mean body weight change (%) of males decreased significantly during the entire treatment period (Day 1-46) at 1000 mg/kg and during the first two weeks of treatment at 556 mg/kg. Males from the recovery group also demonstrated significant decrease in the mean body weight changes during day 1-29. Females gained significantly less weight during gestation (days 1-20), but the mean body weight change did not alter in the recovery group. The feed consumption of all treatment and recovery groups of both sexes were comparable and did not show any significant differences as compared with respective controls. In addition, no treatment related and/or dose dependent changes were noted in haematological and clinical biochemistry parameters up to 1000 mg/kg body weight. External and visceral examination of all treated and control rats including recovery groups did not reveal any abnormality of pathological significance. The absolute and relative organ weights of brain, adrenals, ovaries with oviduct, testes, epididymis, heart, liver, kidney spleen, thymus of dosed rats of either sex did not differ significantly when compared to respective control groups. Oestrus cyclicity was regular i.e. 3-5 days in control, treatment and recovery groups. During cohabitation, all females from treatment and control groups showed evidence of copulation. Most females showed pre-coital interval less than 5 days, except 4 females from the 1000 mg/kg, 1-1 females from 556 mg/kg and control groups which showed pre-coital interval longer than 5 days. Only 8 out of 13 females achieved pregnancy in the high-dose group and the pregnancy/fertility index decreased to 61.54%. The gestation length was comparable in all treatment and control groups. Examination of the uterus content revealed that the number of implantation and corpora lutea did not differ in treated and control rats. The reproductive organs of all dosed rats were subjected to histopathological evaluation. Histopathological lesions observed in males included focal minimal to mild seminiferous tubule degeneration, focal minimal to mild and multifocal minimal retention of mature sperm, focal minimal to mild sloughing of round spermatid/pachytene spermatocyte. Histopathological lesions observed in females included minimal reduction of stromal cells, focal moderate necrosis and nodular hyperplasia of uterus as well as minimal lymphocytic infiltration of the cervix. However, the severity and incidence of histopathological lesions of testis, uterus and cervix were well comparable with respective control groups and exhibited no dose relationship. Hence, the occurrence of these lesions was considered spontaneous or incidental in nature and did not attributed to the test chemical administration. Developmental parameters such as number of live births, litter size, number of alive pups at PND 4, and post-implantation loss were comparable in all groups. Postnatal loss (%) increased at 556 and 1000 mg/kg but it was not statistically significant. The fetal survival index at PND 4 slightly decreased in middle and top dose groups but it did not differ significantly from the control. Pups demonstrated normal growth rate as the body weight at birth and PND 4 and were comparable in treated and control groups. The body weight gain of pups at PND 0-4 were comparable in both control and treated groups. Gross observation of pups revealed no abnormalities attributable to the test chemical administration. Further, terminally sacrifice pups of all treated groups did not reveal any lesion of pathological significance at any dose when compared with control. Pups died during the course of the study did not revealed treatment related extremal or visceral lesions. In summary, parental animals gained significantly less weight when male and female rats were orally administered with 1000 mg/kg of test chemical for 47-54 days. In addition, the prenatal and early postnatal exposure to the test chemical did not evoke adverse effect on neonatal survival, growth and development, when female Wistar rats were orally administered up to 1000 mg/kg for 54 days. Hence, the developmental NOAEL for F1 generation was 1000 mg/kg body weight/day.Similarly, the maternal developmental NOAEL was 1000 mg/kg/day based on the lack of significant alterations of maternal developmental (in utero) parameters examined.

Study 3:

The possible adverse effect of the test chemical on foetus/prenatal development was tested using mated female Sprague-Dawley rats. The test chemical was dissolved in corn oil and administered to 24 rats/dose on the dosage levels of 0 (corn oil), 50, 200, 500 mg/kg/day via oral gavage during gestation days 6-15. Animals were observed for mortality and clinical signs, changes in body weight and food consumption were recorded as well. In utero parameters such as the numbers of total implantations, post-implantation loss, fetal resorption were recorded as well as the litter weight and fetal viability. Approximately 1/2 of the fetuses in each litter were processed for soft-tissue evaluations while the other half for skeletal evaluations. Two deaths occurred at 500 mg/kg. Excessive alopecia, salivation and/or anogenital staining was observed but no pattern of treatment relationship could be determined. Statistically reduced maternal weight gain and food consumption were observed at 200 and 500 mg/kg. The prenatal exposure to the test chemical did not have effect on fetal resorptions, post-implantation loss fetal viability, or total number of implantations. The mean litter weights in treated and control groups were comparable. No significant increases were observed in incidence of malformations or variations at any treatment level. In conclusion, the oral administration of the test chemical to pregnant SD rats was maternally toxic evidenced by the treatment related clinical signs and reduced maternal weight gain and food consumption at 200 and 500 mg/kg. Hence, the NOAEL for systemic toxicity was 50 mg/kg/day. No changes in maternal developmental parameters were observed at any dose level when pregnant SD rats were orally dosed during GD 6-15 and therefore the maternal developmental NOAEL was 500 mg/kg body weight/day. In addition, the prenatal exposure to the test chemical up to 500 mg/kg had no effect on growth and survival of the foetuses, and/or on the incidences in external, skeletal and soft tissue malformations and variations in fetuses and, therefore the developmental NOAEL for was 500 mg/kg body weight/day for the F1 generation.    

Study 4:

A prenatal developmental toxicity study was performed to investigate the toxic effect of the test chemical on fetus organogenesis and development. Groups of 20 pregnant Wistar rats were administered 0, 10, 100, 500, or 1,000 mg/kg bw/day by gavage during gestation days 6-15 (GD 6-15). On day 20 of gestation, pregnancies were terminated, and the fetuses were examined for intrauterine death, and internal, external and skeletal malformations. Maternal parameters included mortality, body weight, food consumption, and clinical and gross examinations. No maternal toxic effects on parameters examined were observed as no death and alterations of body weight, food consumption was reported, and clinical and gross examinations did not reveal any effect attributable to test chemical administration. Examination of the uterus content revealed that the number of implantation and corpora lutea, live/dead fetuses, or resorptions, implantation ratio, sex ratio, or placenta weigh did not differ in treated and control rats.The fetal body weight was significantly decreased at 1000 mg/kg and significantly increased at low and middle doses, thus these alterations were inconclusive and showed no dose-dependence.There was a statistically significant increase in the combined incidence of organ variations (i.e., slight dilatation of the lateral ventricle and renal pelvis, and presence of levo-umbilical artery) in animals from the 500 and 1000 mg/kg dose groups. The only skeletal malformation (fused ribs) was seen in one fetus of the high-dose group, which did not increase the incidence of skeletal malformations compared to controls. The authors suggested that the skeletal malformations were related to the significant decrease in fetal body weight. Skeletal variations (i.e., wavy ribs, dumbbell shaped vertebrae, absence/splitting of thoracic vertebrae, presence of lumbar ribs and degree of ossification) were statistically increased at 1000 mg/kg. However, skeletal variations are minor structural changes which have little or no detrimental effect on the animal and often are transient changes. No increase in intrauterine death or external variations was noted at any dose level.In conclusion, the oral administration of the test chemical up to 1000 mg/kg was not maternally toxic evidenced by the absence of alterations of parameters examined. In addition, the prenatal exposure to 1000 mg/kg of the test substance did not alter the normal growth and development of offspring and, consequently the developmental NOAEL was 1000 mg/kg for the F1 generation.   

Justification for classification or non-classification

Weighting of evidence from several independent studies led to the conclusion that the test chemical exert no adverse effects on male and female fertility and reproductive performance and the pre- and early postnatal exposure to the test chemical does not interfere with the normal growth and development of offspring or increases the incidence of birth defects. Hence, the test chemical is not classified for reproductive and developmental toxicity according to CLP criteria.